1. Field of the Invention
The present invention in the fields of immunology and virology relates to monoclonal antibodies specifically directed to HIV-1 neutralizing epitopes, and particularly human monoclonal antibodies, lymphoblastoid and heterohybridoma cell lines which produce such antibodies, and methods for producing the human monoclonal antibodies and the lymphoblastoid and heterohybridoma cell lines.
2. Description of the Background Art
The human immunodeficiency viruses (HIV) have been implicated as the causative agent of acquired immune deficiency syndrome (AIDS). Two different HIV families have been identified to date: HIV-1 and HIV-2. It is currently believed that the majority of individuals that become infected with HIV eventually will develop AIDS and are likely to succumb to fatal infections and/or malignancies. Currently, it is estimated that approximately 1.5 million persons have been infected by HIV in the United States alone. Thus, treatment and prevention of HIV infection is among the leading public health challenges today. There is no known cure for HIV infection, which invariably progresses to AIDS and subsequently to the death of the infected individual.
Immunotherapeutic approaches to AIDS have been largely limited to vaccination. Vaccine therapy has relied upon virion proteins, expressed by infected cells, that are designated inter alia as p24, gp41, gp120, etc. (See, for example, Essex, U.S. Pat. No. 4,725,6569.) A number of candidate HIV vaccines, including those involving recombinant HIV antigens, have been prepared and tested, but none has yet proven to be of clinical significance (Berman, P. W. et al., Nature 345:622-625 (1990); Girard, M. et al., Proc. Natl. Acad. Sci. USA 88:542-546 (1991); Berzofsky, J. A. FASEB J. 5:2412-2418 (1991)).
Several studies have demonstrated that effective protection against HIV-1 and HIV-2 infection can be achieved by passive immunization. Initially, two studies showed that immunoglobulin preparations from infected chimps or from infected humans could protect from infection with the HTLV-IIIB isolate of HIV-1 (Emini, E. A. et al., J. Virol. 64:3674 (1990); Prince, A. M. et al., AIDS Res. Hum. Retrovir. 7:971 (1991)). Subsequently, a mouse monoclonal antibody (mAb) against the V3.sub.IIIB loop of gp120, was shown to prevent chimpanzee infection with a homologous strain of HIV-1 (Emini et al., supra). The same mAb, after molecular manipulation to replace the mouse constant regions with human constant regions, could also protect chimpanzees against infection when the mAb was administered before exposure and even 10 minutes after virus challenge (Emini et al., supra). Additionally, serum from vaccinated macaques protected other macaques from subsequent challenge with HIV-2/SIV (Putkonen, P. et al., Nature 352:436 (1991)). Studies of active immunization also suggest that neutralizing antibodies to the third variable region (V3) of the HIV-1 envelope glycoprotein, gp120, may prevent infection of chimpanzees (Berman, P. et al., Nature 345:622 (1990) and Girard, M., Proc. Natl. Acad. Sci. USA 88:542 (1991)).
The use of mAbs for treatment of HIV-1 infections has been hampered because most mAbs directed against HIV-1 proteins currently available in therapeutic quantities are of rodent origin. Administration of non-human antibodies to humans can cause dangerous and even life-threatening immunologic reactions. In addition, such rodent mAbs may not be as effective in interacting with human effector cells or effector molecules (such as the complement system).
Stable human cell lines producing HIV-1-specific mAbs, and the mAb products directed against HIV-1 components, are useful for treating and/or diagnosing individuals infected with this virus. However, human mAbs in general, and those directed against HIV in particular, have proven to be extremely difficult to produce.
Among the explanations for this difficulty are:
(a) The most readily available source of lymphocytes from humans, the peripheral blood, normally contains few antibody-producing cells and, in some instances, none at all; PA1 (b) transformation of antibody-producing cells can be achieved using Epstein-Barr virus (EBV), but production is often unstable and the level of antibody produced is often low; PA1 (c) whereas the level and the stability of antibody production can be enhanced by fusion of EBV-transformed human B lymphocyte lines to mouse myeloma cells, such hybrids (called "heteromyelomas") readily lose human chromosomes, and ultimately antibody production as well; and PA1 (d) whereas fusion of normal or transformed B cells to human lymphoblastoid lines or to heteromyelomas can stabilize antibody production, few satisfactory parent lines of this cell type have been available.
Neutralizing antibodies are considered to be essential for protection against viral infection. For this reason, any synthetic vaccine against HIV-1 must include epitopes which induce neutralizing antibodies. Analysis of the reactivity patterns of sera of HIV-infected subjects has revealed that two major types of neutralizing antibodies are elicited by HIV infection. One is directed against the third hypervariable domain (V3) of gp120, and the other against a conserved region of gp120, involved its binding to CD4 (the CD4-binding domain, CD4bd). Antibodies to the V3 loop had been considered to be strain-specific because they appeared to be reactive with only the eliciting strain of HIV (Rusche, J. R. et al., Proc. Natl. Acad. Sci. USA 85:3198 (1988)). It has recently been shown that some anti-V3 antibodies can react with more than one viral strain when the amino acid sequence recognized by the antibody is present in both strains (Javaherian, K. et al., Science 250:1590 (1990)). Antibodies against V3 occur in the early phase of infection, but their protective role may decrease in the course of disease due to the emergence of resistant mutants (Nara, P. et al., J. Virol. 64:3779 (1990)).
Antibodies to the CD-4 binding site are observed in later phases of infection and, because they recognize a conserved region, they may neutralize many (but not all) strains of HIV and thus be "group-specific" (Ho, D. D. et al., J. Virol. 65:489 (1991)). However, anti-V3 antibodies appear to be more potent than antibodies to the CD4bd in terms of their ability to neutralize the virus (Kang, C. et al., Proc. Natl. Acad. Sci. USA 88:6171 (1991)).
The V3 neutralizing epitope of HIV-1 is located between two cysteine residues which participate in an intrachain disulfide bond which is predicted to form a hairpin loop structure in the protein. The "tip" of the loop consists of a sequence of four amino acids, Gly-Pro-Gly-Arg (G-P-G-R) (amino acids 315-318 of the V3 loop of the MN isolate residues 15-18 of (SEQ ID NO:1)) that is essentially conserved in North American and European virus isolates (Goudsmit, J., et al., Proc. Natl. Acad. Sci. USA 85:4478 (1988); LaRosa, G. J., et al., Science 249:932 (1990)) and is flanked by amino acids which differ between various HIV-1 isolates. The V3 loop of gp120 has been shown to be important for biological activity of the virus, including infectivity.
Therapeutic use of anti-HIV antibodies has been proposed (Jackson, G. G, Lancet ii:647 (1988); Chanh, T. C. et al., EMBO J. 5:3065 (1986); Karpas, A. et al., Proc. Natl. Acad. Sci. USA 85:9234 (1988)).
A number of approaches have been taken to preparing antibodies against HIV. For example, Putney et al. (European Patent Publication EP311228) disclosed DNA and proteins useful in assays for detecting and quantifying antibodies against HIV. Putney et al. (European Patent Publication EP255190) disclosed recombinant DNA transfer vectors which comprise all or part of the nucleotide sequence, the translated regions of which encode various fragments of the envelope protein. These protein fragments were said to be useful in immunoassays for detection of HIV antibodies, as antigenic components of AIDS vaccines, and for stimulation of lymphocyte proliferative responses in infected individuals.
Wang (European Patent Publication EP328403) disclosed peptides specifically immunoreactive with antibodies to HIV-1 which neutralize antibodies to HIV-gp120. The disclosed peptides comprise 15-40 amino acids in a sequence corresponding to a region in HIV gp120 which are peptide 126, peptide 127, and analogues thereof. These peptides are used as solid phase immunoadsorbents for detection of antibodies to HIV gp120, including neutralizing antibodies.
Goudsmit et al. (European Patent Publication EP311219) disclosed oligopeptides of 8-17 amino acids in a sequence corresponding to the variable region (V3) in the gp120 protein. Also disclosed are antibodies to the oligopeptides. The oligopeptides comprise the .beta.-turn amino acid sequence GPG or GPGR at positions 312-314 or 312-315 of the IIIB isolate residues 10-12 or 10-13 of (SEQ ID NO:2), and flanking amino acid sequences having a length of at least 1 and preferably at least 2 amino acids and variations in which the GPG or GPGR sequence has been replaced by a different .beta.-turn sequence, and variations in which the free amino group of the N-terminal amino acid and/or the free carboxyl group of the C-terminal amino acid has been blocked or otherwise modified. This document also discusses antibodies to these oligopeptides.
Rusche et al. (European Patent Publication EP306219) disclosed HIV-1 proteins or peptides and DNA sequences coding therefor. These proteins and peptides were said to be useful in the diagnosis, prophylaxis, and therapy of AIDS, in the preparation of HIV vaccines, and for stimulation of lymphocyte proliferative responses in HIV-infected humans.
Petteway et al. (PCT Publication WO 8805824) disclosed a method for producing and selecting a hybridoma cell which produces a mAb to a viral glycoprotein, such as an HIV glycoprotein. The method is said to be useful for obtaining mAbs to HIV proteins such as gp120 and gp41.
Kennedy et al. (European Patent Publication EP245362) disclosed a synthetic peptide which induced an immune response to the virus causing AIDS and ARC. This peptide has a sequence homologous to a portion of the amino acid sequence of the gp120 or gp41 envelope glycoproteins of HIV. The peptides are said to be useful to vaccinate against viral causative agents of AIDS and in diagnostic assays for AIDS.
Grunow et al., (J. Immunol. Meth. 106:257-265 (1988)) described the construction a heteromyeloma cell line designated CB-57 by fusing normal human B lymphocytes and a murine myeloma. This heteromyeloma was then used for fusion with EBV-transformed B cells to obtain three cloned heterohybridomas which showed about 30-fold enhanced secretion of IgM antibodies relative to the EBV-transformed parent line. CB-57 cells were also fused with PBL from HIV seropositive individuals, resulting in four heterohybridomas producing IgM anti-p25 antibodies or IgG.sub.1 antibodies to p25 or gp41. No anti-gp120 heterohybridomas were reported. A later report from the same group (Berzow, D. et al., Proc. IVth Int'l. AIDS Conf., Montreal, 1989, abstr. TCP 150) indicated that one clone was capable of continuous production of antibodies to gp120, but no data were available on the activity of this antibody.
Scott, C. F. et al. (Proc. Natl. Acad. Sci. USA 87:8597-8601 (1990 Nov.)) disclosed a human IgG.sub.1 mAb termed N701.9b which was derived from EBV-transformed B cells of an HIV-infected donor. This antibody recognized the principal neutralizing domain within the V3 loop of MN-like strains, more particularly an epitope containing at least 7 amino acids in positions 316-322. This antibody had neutralizing activity for MN but not IIIB or RF strains of HIV-1. This antibody was therefore of the "type-specific" variety, in stark contrast to some of the monoclonal antibodies of the present invention which have broad reactivity against diverse HIV-1 strains.
A broadly neutralizing murine mAb NM-01 that recognizes the V3 loop (residues 312-326) of HIV-1 was disclosed by Ohno, T. et al. (Proc. Natl. Acad. Sci. USA 88:10726-10729 (1991 Dec.)). This antibody could neutralize both MN and IIIB strains and reacted equally with loop peptides from MN, IIIB, RF and CDC4 isolates. This antibody reacted less well, or not at all, with loop peptides from NY5, Z2, Z6 and ELI isolates. The authors concluded that this broad reactivity was due to recognition of the GPGR sequence (residues 15-18 of SEQ ID NO:1). This murine antibody is clearly distinct from the human mAbs disclosed in the present application and is highly limited in its clinical utility.
Other murine mAbs which are broadly reactive to some extent are described in .ANG.kerblom, L. et al., AIDS, 4:953-960 (1990). The neutralizing capacity of such mAbs appeared to be limited to a sequence QRGPGR (residues 10-15 of SEQ ID NO:2) of the HTLV-III.beta. strain.